Hexokinase Method

Excerpt

Glucose is an aldose-monosaccharide with the chemical formula of CHO. Humans derive glucose by consuming food of plant or animal origin. Complex carbohydrates are digested in the gastrointestinal tract, converted to simple monosaccharides, and absorbed. Digestion of milk (lactose) and table sugar (sucrose) disaccharides yields galactose and fructose, respectively. These monosaccharides can also be converted to glucose during body metabolism. Besides diet, glucose can be generated by breaking down liver glycogen (glycogenolysis) and non-carbohydrate sources via gluconeogenesis.

Plasma glucose level is meticulously regulated within a defined range. This is regulated by insulin and glucagon hormones. Insulin is secreted in response to high plasma glucose levels after meals. Insulin increases glycolysis, glycogenesis, lipogenesis, and protein anabolism and decreases glycogenolysis, gluconeogenesis, lipolysis, and protein catabolism. Glucagon is secreted in response to low plasma glucose levels, usually during fasting. Glucagon increases plasma glucose levels by increasing glycogenolysis and gluconeogenesis. The hormone decreases glycogenesis and glycolysis. Catecholamines, glucocorticoids, and thyroid hormones also increase plasma glucose levels.

Any pathology that disturbs the balance of these regulatory mechanisms can alter the plasma glucose level. Plasma glucose is commonly ordered to evaluate the fluctuation in these mechanisms. Plasma glucose levels measured in different physiological scenarios, such as fasting, and 2-hour post-prandial are utilized to screen, diagnose, and monitor pathological conditions.

Many point-of-care testing devices are utilized in emergency and home care settings, which use capillary blood to estimate plasma glucose via a biosensor-based chip. The major drawbacks of such devices are imperfection in dispensing the blood sample to the biosensor chip, interference by tissue interstitial fluid, and lack of timely calibration and quality control. Various chemical and enzymatic methods are available for plasma glucose estimation. Due to higher time requirements and relatively less accuracy and precision than enzymatic methods, chemical methods such as Folin Wu and O-Toluidine are less utilized in clinical laboratories. In the hospital’s clinical laboratory, enzymatic methods based on glucose oxidase-peroxidase (GOD-POD) method, glucose dehydrogenase (GDH) method, and hexokinase (HK) method are commonly utilized.

In the GOD-POD method, β-D-glucose is first oxidized by the GOD enzyme, leading to hydrogen peroxide (H₂O₂) formation. This H₂O₂ reacts with a colorless chromogen substrate in the presence of peroxidase (POD) to produce a colored product. The intensity of color is proportional to the glucose concentration in the sample. The GOD enzyme can act on β-D-glucose only. At equilibrium, alpha and β isomers of D-glucose are at 36% and 64%, respectively. To optimize the results, a mutarotase enzyme should be added, or an extended incubation time is needed to complete the reaction.

In the GDH method, the GDH enzyme acts on beta-D-glucose and NAD+ (nicotinamide adenine dinucleotide) to form gluconolactone and NADH (dihydronicotinamide adenine dinucleotide). The amount of generated NADH is proportional to the glucose concentration in the sample. This method also requires mutarotase or extended incubation time to complete the reaction.

Many POCT analyzers, including blood-gas instruments, measure glucose concentrations using the glucose oxidase method. Urine dipsticks are widely used to screen for glucose in the urine. All strips use glucose oxidase with a chromogenic assay. The hexokinase method is an exact and accurate method for plasma glucose estimation. Serum or plasma is first deproteinized by barium hydroxide, and zinc sulfate and clear supernatant are used for the reaction. Glucose in the sample first reacts with adenosine triphosphate (ATP) with the help of hexokinase to form glucose-6-phosphate. Glucose-6-phosphate is then catalyzed by glucose-6-phosphate dehydrogenase (G6PD) in the presence of NADP+ (nicotinamide adenine dinucleotide phosphate) or NAD+ to form NADPH (dihydronicotinamide-adenine dinucleotide phosphate) or NADH and 6-phosphogluconate. The amount of NADPH or NADH generated is measured by recording the absorbance at 340 nm, which is proportional to the glucose concentration in the sample.

The cofactor utilized in the G6PD reaction depends upon the origin of the enzyme. If G6PD of leuconostoc mesenteroides bacteria is used, NAD+ is a cofactor. Meanwhile, G6PD from yeast or higher plants requires NADP+ as a cofactor. The advantage of using the bacterial enzyme is that red blood cell G6PD and 6-phosphogluconate dehydrogenase, which use NADP as a substrate, do not interfere with glucose analysis. This reduces the interference in the assay resulting from hemolysis.

Due to the high accuracy and precision, this method is the reference for plasma glucose estimation. The need for deproteinization increases the turnaround time for glucose estimation. The deproteinization step is not performed to minimize the turnaround time in the hospital clinical laboratory, and plasma or serum is directly added to the reagents per assay protocol. The effect of interfering substances in plasma or serum can be nullified by sample blanking. This eliminates the error due to any interfering substance in plasma or serum, which can absorb 340 nm radiation and affect the final result.

The definitive method for glucose determination is isotope dilution mass spectrometry (ID-MS). This technique measures the analyte concentration with the greatest accuracy, so the systematic errors are negligible, and there is a high level of precision with the coefficient of variation (CV) < ±0.5%. External quality assurance programs use this technique to set the target values for lyophilized sera.